Rho-like GTPases are important players in vascular function due to their ability to regulate the actin cytoskeleton. They are involved in physiological processes such as smooth muscle cell contraction, endothelial permeability, platelet activation, leukocyte migration, angiogenesis and wound healing. Moreover, deregulation of Rho GTPases promotes vascular disorders associated with vascular remodeling, altered cell contractility and cell migration such as vascular hyperpermeability, tumor cell invasion, platelet aggregation, atherosclerosis and restenosis and cardiac hypertrophy. The primary objective of this proposal is to investigate the role of thiol modification in regulation of redox active Rho GTPases. We have previously demonstrated that a subset of Rho GTPases, i.e., Rac1, RhoA and Cdc42, contain a thiol in the guanine nucleotide binding site that reacts with reactive oxygen and nitrogen species (RNS, ROS) to regulate Rho GTPase activity. Specifically, guanine nucleotide binding is modulated by redox agents to promote formation of thiol radical intermediates that facilitate guanine nucleotide oxidation and release of guanine nucleotide substrates. This mechanism is similar to that described by us previously for redox regulation of Ras GTPases. However, in contrast to Ras, we provide evidence that Rho GTPases are also regulated by two-electron oxidative mechanisms (ionic), in addition to the radical mediated mechanism of guanine nucleotide dissociation, due to the location of the reactive cysteine in the conserved phosphoryl binding loop. In this proposal, we seek to investigate the role of thiol oxidation in regulating the structure, biochemical and cellular activity of Rac1 and RhoA GTPases. Rac1 and RhoA have recently been shown to regulate endothelial barrier function in response to hypoxia and ischemia/reoxygenation via remodeling of the actin cytoskeleton and adherens junctions in a ROS-dependent manner. The primary function of the endothelial lining of blood vessels is to maintain a selective permeability barrier between blood and tissues, and breakdown of endothelial barrier function induced by hypoxia has been shown to contribute to lung diseases such as acute respiratory distress syndrome and ischemia-reperfusion injury. Thus, information derived from this effort may aid in developing therapies to combat vascular pathologies such as respiratory distress syndrome and ischemia-reperfusion injury.